Unknown,Transcriptomics,Genomics,Proteomics

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Gene expression profiling of Calu-6 lung cancer cells transfected with scrambled siRNA and LAPTM4B-specific siRNA

ABSTRACT: We recently characterized the adjacent airway field of cancerization in NSCLC by whole transcriptome expression analysis and demonstrated that lysosomal protein transmembrane 4 beta (LAPTM4B) was an elevated field cancerization marker in NSCLCs and in adjacent but not in distant normal-appearing airways. We also found that LAPTM4B was up-regulated in NSCLCs compared to normal lung and promoted anchorage-dependent growth of lung cancer cells. Previous reports suggested that LAPTM4B is activated following metabolic and genotixc stress. The precise role of LAPTM4B in lung cancer cell survival and NSCLC pathogenesis is still elusive. In the present study we sought to examine how LAPTM4B expression levels impact downstream expression profiles and cell signaling in order to gain better insights into the function of this putative oncogene in lung cancer. Calu-6 lung cancer cells were transfected with SMARTpool (Dharmacon)control/scrambled siRNA or siRNA targeting LAPTM4B. Following transfection, cells were cultured in the presence or absence of 10% fetal bovine serum (FBS). Three independent transfections for each condition were performed representing three biological replicates per condition (total number of samples = 12). RNA interference-mediated knock-down of LAPTM4B in samples was confirmed by quantitative real-time PCR. Total RNA was profiled using the Human Gene 1.0 ST platform.

ORGANISM(S): Homo sapiens

SUBMITTER: Humam Kadara

PROVIDER: E-GEOD-66606 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Epigenetic changes largely contribute to the regulation of gene expression in cancer cells. DNA methylation is part of the epigenetic gene regulation complex which is relevant for the pathogenesis of cancer. We performed a genome-wide search for methylated CpG islands in tumors and corresponding non-malignant lung tissue samples of 101 stage I-III non-small cell lung cancer (NSCLC) patients by combining methylated DNA immunoprecipitation and microarray analysis using NimbleGenM-BM-4s 385K Human CpG Island plus Promoter arrays. By testing for differences in methylation between tumors and corresponding non-malignant lung tissues, we identified 298 tumor-specifically methylated genes. From many of these genes epigenetic regulation was unknown so far. Gene Ontology analysis revealed an over-representation of genes involved in regulation of gene expression and cell adhesion. Expression of 182 of 298 genes was found to be upregulated after 5-aza-2M-BM-4-deoxycytidine (Aza-dC) and/or trichostatin A (TSA) treatment of 3 NSCLC cell lines by Affymetrix microarray analysis. In addition, methylation of selected genes in primary NSCLCs and corresponding non-malignant lung tissue samples were analyzed by methylation-sensitive high resolution melting analysis (MS-HRM). Our results obtained by MS-HRM analysis confirmed our data obtained by MeDIP-chip analysis. Moreover, by comparing methylation results from MeDIP-chip analysis with clinico-pathological parameters of the patients we observed methylation of HOXA2 as potential parameter for shorter disease-free survival of NSCLC patients. In conclusion, using a genome-wide approach we identified a large number of tumor-specifically methylated genes in NSCLC patients. Our results stress the importance of DNA methylation for the pathogenesis of NSCLCs. Overall, samples of 3 untreated, with Aza-dC treated and with Aza-dC/TSA treated NSCLC cell lines were hybridized to Affymetrix HG-U133_plus_2.0 microarrays (18 in total).

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Gene expression profiling of Calu-6 lung cancer cells transfected with scrambled siRNA and LAPTM4B-specific siRNA

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